Integrin-mediated interactions with extracellular matrix proteins for nucleus pulposus cells of the human intervertebral disc.

The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin-mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM-111 and LM-511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM-binding integrins was quantified for human NP cells using flow cytometry. The cell-ECM attachment mechanism was determined by quantifying cell attachment to LM-111, LM-511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3, and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM-111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival, and biosynthesis in cell-based therapeutics.

Expansion of the alpha 2-adrenergic receptor family: cloning and characterization of a human alpha 2-adrenergic receptor subtype, the gene for which is located on chromosome 2.

Pharmacologic, biochemical, and genetic analyses have demonstrated the existence of multiple alpha 2-adrenergic receptor (alpha 2AR) subtypes. We have cloned a human alpha 2AR by using the polymerase chain reaction with oligonucleotide primers homologous to conserved regions of the previously cloned alpha 2ARs, the genes for which are located on human chromosomes 4 (C4) and 10 (C10). The deduced amino acid sequence encodes a protein of 450 amino acids whose putative topology is similar to that of the family of guanine nucleotide-binding protein-coupled receptors, but whose structure most closely resembles that of the alpha 2ARs. Competition curve analysis of the binding properties of the receptor expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 2AR pharmacology. Hybridization with somatic cell hybrids shows that the gene for this receptor is located on chromosome 2. Northern blot analysis of various rat tissues shows expression in liver and kidney. The unique pharmacology and tissue localization of this receptor suggest that this is an alpha 2AR subtype not previously identified by classical pharmacological or ligand binding approaches.

Involvement of tyrosine residues located in the carboxyl tail of the human beta 2-adrenergic receptor in agonist-induced down-regulation of the receptor.

Chronic exposure of various cell types to adrenergic agonists leads to a decrease in cell surface beta 2-adrenergic receptor (beta 2AR) number. Sequestration of the receptor away from the cell surface as well as a down-regulation of the total number of cellular receptors are believed to contribute to this agonist-mediated regulation of receptor number. However, the molecular mechanisms underlying these phenomena are not well characterized. Recently, tyrosine residues located in the cytoplasmic tails of several membrane receptors, such as the low density lipoprotein and mannose-6-phosphate receptors, have been suggested as playing an important role in the agonist-induced internalization of these receptors. Accordingly, we assessed the potential role of two tyrosine residues in the carboxyl tail of the human beta 2AR in agonist-induced sequestration and down-regulation of the receptor. Tyr-350 and Tyr-354 of the human beta 2AR were replaced with alanine residues by site-directed mutagenesis and both wild-type and mutant beta 2AR were stably expressed in transformed Chinese hamster fibroblasts. The mutation dramatically decreased the ability of the beta 2AR to undergo isoproterenol-induced down-regulation. However, the substitution of Tyr-350 and Tyr-354 did not affect agonist-induced sequestration of the receptor. These results suggest that tyrosine residues in the cytoplasmic tail of human beta 2AR are crucial determinants involved in its down-regulation.

Cloning and expression of a human kidney cDNA for an alpha 2-adrenergic receptor subtype.

An alpha 2-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet alpha 2-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet alpha 2-adrenergic receptor and is consistent with the structure of other members of the family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the alpha 2-adrenergic ligand [3H]rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the alpha 2B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet alpha 2-adrenergic receptor (alpha 2A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective alpha-adrenergic ligands.

Inactivation of the von Hippel-Lindau tumor suppressor leads to selective expression of a human endogenous retrovirus in kidney cancer.

A human endogenous retrovirus type E (HERV-E) was recently found to be selectively expressed in most renal cell carcinomas (RCCs). Importantly, antigens derived from this provirus are immunogenic, stimulating cytotoxic T cells that kill RCC cells in vitro and in vivo. Here, we show HERV-E expression is restricted to the clear cell subtype of RCC (ccRCC) characterized by an inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene with subsequent stabilization of hypoxia-inducible transcription factors (HIFs)-1α and -2α. HERV-E expression in ccRCC linearly correlated with HIF-2α levels and could be silenced in tumor cells by either transfection of normal VHL or small interfering RNA inhibition of HIF-2α. Using chromatin immunoprecipitation, we demonstrated that HIF-2α can serve as transcriptional factor for HERV-E by binding with HIF response element (HRE) localized in the proviral 5' long terminal repeat (LTR). Remarkably, the LTR was found to be hypomethylated only in HERV-E-expressing ccRCC while other tumors and normal tissues possessed a hypermethylated LTR preventing proviral expression. Taken altogether, these findings provide the first evidence that inactivation of a tumor suppressor gene can result in aberrant proviral expression in a human tumor and give insights needed for translational research aimed at boosting human immunity against antigenic components of this HERV-E.

Contributions Of the Human Medial Prefrontal Cortex To Associative Recognition Memory: Evidence From Functional Neuroimaging

Neuroimaging studies of episodic memory, or memory of events from our personal past, have predominantly focused their attention on medial temporal lobe (MTL). There is growing acknowledgement however, from the cognitive neuroscience of memory literature, that regions outside the MTL can support episodic memory processes. The medial prefrontal cortex is one such region garnering increasing interest from researchers. Using behavioral and functional magnetic resonance imaging measures, over two studies, this thesis provides evidence of a mnemonic role of the medial PFC. In the first study, participants were scanned while judging the extent to which they agreed or disagreed with the sociopolitical views of unfamiliar individuals. Behavioral tests of associative recognition revealed that participants remembered with high confidence viewpoints previously linked with judgments of strong agreement/disagreement. Neurally, the medial PFC mediated the interaction between high-confidence associative recognition memory and beliefs associated with strong agree/disagree judgments. In an effort to generalize this finding to well-established associative information, in the second study, we investigated associative recognition memory for real-world concepts. Object-scene pairs congruent or incongruent with a preexisting schema were presented to participants in a cued-recall paradigm. Behavioral tests of conceptual and perceptual recognition revealed memory enhancements arising from strong resonance between presented pairs and preexisting schemas. Neurally, the medial PFC tracked increases in visual recall of schema-congruent pairs whereas the MTL tracked increases in visual recall of schema-incongruent pairs. Additionally, ventral areas of the medial PFC tracked conceptual components of visual recall specifically for schema-congruent pairs. These findings are consistent with a recent theoretical proposal of medial PFC contributions to memory for schema-related content. Collectively, these studies provide evidence of a role for the medial PFC in associative recognition memory persisting for associative information deployed in our daily social interactions and for those associations formed over multiple learning episodes. Additionally, this set of findings advance our understanding of the cognitive contributions of the medial PFC beyond its canonical role in processes underlying social cognition.

Influence of Increased Human Presence in the Mills River Basin on Water Availability and Drought

Periods of drought and low streamflow can have profound impacts on both human and natural systems. People depend on a reliable source of water for numerous reasons including potable water supply and to produce economic value through agriculture or energy production. Aquatic ecosystems depend on water in addition to the economic benefits they provide to society through ecosystem services. Given that periods of low streamflow may become more extreme and frequent in the future, it is important to study the factors that control water availability during these times. In the absence of precipitation the slower hydrological response of groundwater systems will play an amplified role in water supply. Understanding the variability of the fraction of streamflow contribution from baseflow or groundwater during periods of drought provides insight into what future water availability may look like and how it can best be managed. The Mills River Basin in North Carolina is chosen as a case-study to test this understanding. First, obtaining a physically meaningful estimation of baseflow from USGS streamflow data via computerized hydrograph analysis techniques is carried out. Then applying a method of time series analysis including wavelet analysis can highlight signals of non-stationarity and evaluate the changes in variance required to better understand the natural variability of baseflow and low flows. In addition to natural variability, human influence must be taken into account in order to accurately assess how the combined system reacts to periods of low flow. Defining a combined demand that consists of both natural and human demand allows us to be more rigorous in assessing the level of sustainable use of a shared resource, in this case water. The analysis of baseflow variability can differ based on regional location and local hydrogeology, but it was found that baseflow varies from multiyear scales such as those associated with ENSO (3.5, 7 years) up to multi decadal time scales, but with most of the contributing variance coming from decadal or multiyear scales. It was also found that the behavior of baseflow and subsequently water availability depends a great deal on overall precipitation, the tracks of hurricanes or tropical storms and associated climate indices, as well as physiography and hydrogeology. Evaluating and utilizing the Duke Combined Hydrology Model (DCHM), reasonably accurate estimates of streamflow during periods of low flow were obtained in part due to the model’s ability to capture subsurface processes. Being able to accurately simulate streamflow levels and subsurface interactions during periods of drought can be very valuable to water suppliers, decision makers, and ultimately impact citizens. Knowledge of future droughts and periods of low flow in addition to tracking customer demand will allow for better management practices on the part of water suppliers such as knowing when they should withdraw more water during a surplus so that the level of stress on the system is minimized when there is not ample water supply.